Method for the detection of influenza a/b viruses

ABSTRACT

The invention concerns a method for the detection of an infection with influenza A and/or influenza B virus comprising the steps: i) obtaining a saliva sample, ii) preparing the saliva sample for a detection reaction and iii) detecting the influenza A and/or influenza B virus in the saliva sample. The invention in addition concerns a test kit for the detection of an infection by the influenza A and/or influenza B virus containing: i) a device for the collection of a saliva sample and ii) reagents and auxiliary agents for the detection of influenza A and/or influenza B viruses. Furthermore the invention concerns the use of saliva as a sample material for the detection of an infection with the influenza A and/or influenza B virus.

[0001] The invention concerns a method for the detection of influenzaA/B viruses, a corresponding test kit and the use of saliva as a samplematerial for the detection of influenza A/B viruses.

[0002] Influenza is a frequently underestimated infectious disease whichcan result in high morbidity and mortality rates especially in elderlypersons and in high-risk patients. Influenza A and/or influenza Bviruses (also abbreviated influenza A/B viruses in the following) areresponsible for genuine virus influenza which is contracted by several100 million persons worldwide each year. The influenza A and B virusesprimarily infect the nasopharyngeal and oropharyngeal cavities andinitially cause general respiratory symptoms in the affected persons.

[0003] It is not possible even for experienced medical professionals tovery reliably diagnose influenza solely on the basis of the patient'sclinical symptoms since other viruses which infect the nasal orpharyngeal cavity such as adenoviruses, parainfluenza viruses orrespiratory syncitial viruses (RS viruses) cause similar symptoms.

[0004] Due to the high medical importance of influenza infections (flu),almost every country in the world now has a nationally organizedinfluenza monitoring system. For this scheme general practitionersremove swabs from the nose and/or throat and send them to the respectivenational reference centre. The influenza A/B viruses are then usuallydetected by eluting the swabs and subsequently culturing the patientspecimens on mammalian cells such as MDCK cells (Madine-Darby CanineKidney cells).

[0005] The culture in these special laboratories can take up to 14 daysand is thus not of immediate relevance for the diagnosis of theindividual patients. Rather the goal of the national reference centresis to type and subtype the cultured viruses and to report the results tothe World Health Organisation (WHO). The job of the vaccine manufactureris then to adapt next year's influenza vaccines to the latestcirculating viral strains on the basis of the annual WHO recommendation.

[0006] The reason for the high degree of genetic and hence immunologicalvariability especially of the influenza A viruses is due to the factthat a genetic shift (reassortment of the viral genes) can also occur inrare cases in addition to the usual genetic drift (point mutation). Thisis due to the fact that, in contrast to other viruses, the genome of theinfluenza viruses is segmented and that influenza A is a pathogen inhumans as well as in animals.

[0007] The immunodominant antigens present on the surface of the virusare haemagglutin (H) and neuraminidase (N). At present 15 subtypes ofhaemagglutin (H1—H15) and 9 subtypes of neuraminidase (N1—N9) are knownfor influenza A.

[0008] If for example a host (e.g. a pig) is coinfected with aninfluenza virus of the A type which is pathogenic for man and with aninfluenza A bird virus this can result in a reassortment of the viralgenome to form a new influenza A virus subtype which then completelyevades the human immune system when it is transferred back to humans.The most recent example of this was the occurrence of the so-calledavian virus influenza in May 1997 in Hong Kong (type A/H5N1/HongKong/156/97) in which 6 of the 18 affected patients died despite anearly detection and intensive medical care.

[0009] However, in order to cause a larger epidemic or even a worldwidepandemic, a new influenza virus subtype must be transmitted directlyfrom human to human as was the case in 1918/1919 for the firstoccurrence of the subtype H1N1 (Spanish flu, worldwide ca. 50 milliondeaths), 1957 (H2N2, Asian flu, ca. 1 million victims) and 1968 (H3N2Hong Kong flu, ca. 1 million victims).

[0010] A new generation of influenza agents has recently becomeavailable, the so-called neuraminidase inhibitors which for the firsttime allow a causal treatment of influenza and are thus regarded amongexperts as a true breakthrough in the treatment of influenza. Clinicalstudies carried out for the registration of this new class of substanceshowed that successful treatment primarily depends on an early start totreatment after the first clinical symptoms occur. Hence in view of thisnew therapeutic option, the necessity to begin the treatment at an earlystage and the relatively unspecific clinical symptoms, a rapidindividual diagnosis would be helpful as a basis for treatmentdecisions.

[0011] As a result several diagnostic manufacturers have recentlydeveloped rapid immunological influenza tests based on antigen detectionand these rapid tests use nasal and/or throat swabs or liquid obtainedby nasal irrigation as the sample material. Examples of such rapidinfluenza tests are the Influenza A/B Rapid Test from Roche DiagnosticsGmbH, Quick Vue from Quidel and AB FLU OIA from Biostar.

[0012] A particular problem in collecting samples is to remove samplematerial from the affected region of the nasal and pharyngeal cavitywhich contains sufficient amounts of influenza A and/or influenza Bviruses (abbreviated to influenza A/B viruses in the following). Hencethe quality of sampling has a direct influence on the positivity rate ofthe rapid tests which usually have a clinical sensitivity of about 70%i.e. the immunological rapid test is also positive in 70% of all samplesthat are assessed to be influenza positive by the reference method (cellculture). Hence the diagnostic manufacturers point out in the packageinserts for their influenza rapid tests that the specimens should onlybe collected by specially trained medical personnel in order to ensurethat the specimen material obtained does in fact contain adequateamounts of influenza A/B viruses.

[0013] The situation is further complicated by the fact that the regionsin the pharyngeal cavity that have to be swabbed (posterior pharyngealwall, pharynx, tonsils) can indeed differ depending on the viralinfection that is present. This for example means that a swab has to betaken of other regions of the throat in order to detect a streptococcalinfection than for example to detect influenza A/B viruses.

[0014] Taking a nose/throat swab is unpleasant for patients and aparticular problem in the case of (small) children. On the other handchildren are the main carriers of virus influenza at least in the earlyphase of an influenza epidemic due to their social contacts(kindergarten, school) and the fact that their immune system is oftennot yet fully developed.

[0015] Since nose and throat swabs are not a homogeneous samplematerial, the positivity rate of an influenza test is, in addition tothe quality of the swab, also determined by the correct elution i.e. thetransfer of the swab material to a liquid phase which then serves as theactual sample material in the subsequent test.

[0016] Especially the new therapeutic option (neuraminidase inhibitors)will in future result in an increasing need for flu virus tests(influenza A/B viruses) for general practitioners or the patients whichis not compatible with the previously used sample material (swab, nasalirrigation) that requires that specimens be taken by trained (medical)personnel.

[0017] The object of the present invention is to eliminate thedisadvantages of the prior art. In particular the object of theinvention is to provide a method for detecting an influenza A and/orinfluenza B virus infection which can be carried out by untrainedpersonnel or ideally by the patients themselves. Above all it is anobject of the invention to find a sample material from which influenza Aand/or influenza B viruses can be reliably detected which is preferablyhomogeneous and can be collected by untrained personnel or ideally bythe patient themselves in a simple and uncomplicated manner.

[0018] This object is achieved by the subject matter of the invention ascharacterized in the independent patent claims. Preferred embodiments ofthe invention are stated in the dependent claims.

[0019] The invention concerns a method for the detection of an infectionwith the influenza A and/or influenza B virus comprising the steps

[0020] i) obtaining a saliva sample from the individual to be examined

[0021] ii) preparing the saliva sample for the detection or a detectionreaction and

[0022] iii) detecting the influenza A and/or influenza B virus in thesaliva sample.

[0023] The invention additionally concerns a test kit for detecting aninfection with the influenza A and/or influenza B virus containing

[0024] i) a device for collecting a saliva sample and

[0025] ii) reagents and auxiliary agents for the detection of influenzaA and/or influenza B viruses.

[0026] Finally the invention concerns the use of saliva as a samplematerial to detect an infection with the influenza A and/or influenza Bvirus.

[0027] It was surprisingly found that it is possible to reliably detectinfluenza A/B viruses from saliva as a sample material using establisheddiagnostic methods.

[0028] In particular it is surprising that in a preferred embodiment ofthe invention it is possible to detect influenza A/B viruses from salivawithout having to enrich the viruses in the saliva sample when taking asample, but rather spontaneously formed saliva is adequate as a samplematerial for detecting influenza A/B viruses. Spontaneously formedsaliva is understood in the present invention to mean that in order todetect influenza A/B viruses, the influenza A/B viruses are not enrichedin the sample when the saliva sample is collected. Only the saliva thatis present spontaneously in the mouth cavity is collected and forexample transferred to a saliva collecting vessel (e.g. by spittingetc.). It is also possible to collect the saliva sample in the mouthcavity with the aid of an absorbent material such as a cotton fleece(swab) or to use saliva collecting devices familiar to a person skilledin the art (such as the so-called Salivettes from Sarstedt or theOraSure Specimen Collection Device from Epitope Inc. Beaverton, Oreg.97008, USA) to collect the saliva sample.

[0029] Of course it is also possible according to the invention to usesaliva as a sample material for the detection of influenza A/B virusesin which an enrichment (concentration) of the viruses has occurredduring the sampling or in subsequent processing steps.

[0030] In contrast to the previously known sample materials in influenzadiagnostics i.e. nose/throat swabs and liquid obtained by nasalirrigation, the saliva sample material according to the invention andthe simple manner of sample collection allows, a standardized samplecollection by untrained persons or by the patients themselves and thusincreases the diagnostic reliability of influenza A/B virus tests.Moreover saliva is a more homogeneous sample material than the otherpreviously used materials which also contributes to the diagnosticreliability.

[0031] In the method according to the invention a saliva sample isfirstly collected. This can for example be carried out as alreadydescribed above by spitting into a vessel, absorbing a saliva sample bymeans of an absorbent material such as a cotton swab or similarmaterials in the oral cavity or by using a conventional salivacollecting device.

[0032] The saliva sample is subsequently prepared for the detectionreaction in accordance with the detection method to be used(immunological detection or detection by means of nucleic acids).

[0033] For the immunological detection the viral nucleoprotein ofinfluenza A/B viruses is for example released from the saliva sample bylysing reagents. Such reagents are known to a person skilled in the artand can for example contain salts or detergents as the active componentsfor the lysis. Lysing reagents for the detection of influenza virusespreferably contain a detergent (for example Triton® X100, Tween® 20 orbeta-octylglycopyranoside have proven to be suitable), a mild reducingagent (for example N-acetyl L-cysteine or DTT=dithiothreitol),physiological saline (i.e. 0.9% by weight NaCl in 20-50 mM phosphatebuffer), a preservative (for example 0.09% by weight NaN₃) andoptionally a protein to reduce unspecific binding (for exampleBSA=bovine serum albumin or BPLA=bovine plasma albumin). In addition tothe viral nucleoprotein, the immunological test can for example alsodetect the viral matrix protein or the viral polymerases. It is alsopossible to detect the haemagglutinin or the neuraminidase of theinfluenza A/B viruses in which case no lysis is required since theseviral components are present on the viral surface.

[0034] If the detection is based on a nucleic acid, a viral nucleic acid(RNA) is for example isolated, purified and appropriately amplified inthe presence of the primers required for the detection, preferably bymeans of the reverse transcriptase polymerase chain reaction (RT-PCR).These steps are known to a person skilled in the art as are nucleic acidtests without prior amplification.

[0035] The influenza A/B viruses are detected in the saliva sample byknown methods.

[0036] In the case of an immunological detection of for example theviral nucleoprotein, a sandwich complex can be formed usingappropriately labelled antibodies and detected. Competitive test formatsare of course also possible. Although the detection is preferably bymeans of an immunochromatographic test strip that can be visuallyevaluated as a rapid test, the detection can be carried out by means ofall conventional immunological methods, for example ELISA, agglutinationtests, turbidimetric tests etc.

[0037] If the influenza A/B viruses are detected by means of nucleicacids such as RNA, the product of the RT-PCR is preferably labelled bymeans of a labelled primer and the label on the primer (e.g. an enzymeor fluorescent label) is detected according to the type of label.

[0038] A test kit for carrying out the method according to the inventionis also a subject matter of the invention. The test kit contains as animportant component firstly a device for collecting a saliva sample suchas a vessel for spitting into, one or more absorbent cotton swabs or aconventional saliva collection device e.g. a Salivette from the SarstedtCompany or an OraSure specimen collection device from the EpitopeCompany. In addition the test kit contains all reagents and auxiliaryreagents required to detect the influenza A/B viruses in the salivasample. In the case of an immunological detection, these are for examplea lysing buffer to release the viral nucleoproteins, optionally labelledantibodies and optionally a reaction medium (test strips, microtitreplate, test tubes) for carrying out the immunological detection. Thecorresponding reagents and auxiliary agents are known to a personskilled in the art in numerous embodiments. The same applies to thenucleic acid test. In this case the kit for example contains therequired PCR reagents and reaction vessels.

[0039] The invention is further elucidated by the following example:

EXAMPLE

[0040] 1. Detection of an Influenza Virus Infection from Saliva as theSample Material

[0041] In order to demonstrate the suitability of saliva as a samplematerial for detecting influenza viruses, two throat swabs and onesaliva sample were taken from 10 persons suspected to have an influenzainfection and 4 persons which were not suspected of having influenza.The sample material obtained from the throat swabs was collected forcomparative purposes and examined for an influenza A/B virus infection.The results of these investigations were compared with the resultsobtained using saliva as the sample material. Table 1 which is shown atthe end of the experimental part of the example shows an overview of thecomparative results.

[0042] 1.1 Sample Collection

[0043] 1.1.1. Throat Swabs

[0044] The throat swabs were taken using sterile disposable cotton swabsfrom the Copan Italia Company (I-25125 Brescia; order No. 167CS01) inthe manner familiar to a person skilled in the art. The two throat swabstaken from each patient were obtained by swabbing the throat once with aswab having two cotton pads located directly adjacent to one another.This ensures that the two swabs are substantially comparable.

[0045] 1.1.2. Saliva Sample

[0046] The saliva sample was collected by the patients themselves bycollecting ca. 0.5 ml spontaneously formed saliva in a small disposableplastic tube with a screw cap (order No. 62.559.001 from the SarstedtCompany). The saliva collecting methods and devices known to a personskilled in the art (such as Salivettes from the Sarstedt Company or theOraSure specimen collection device from Epitope Inc, Beaverton, Oreg.97008, USA) were intentionally not used in order to demonstrate that itis possible to detect influenza viruses without prior concentration orsaliva processing. Of course it is equally possible according to theinvention to use such saliva collecting methods and devices.

[0047] 1.2 Detection of Influenza A/B Viruses from Throat Swabs (Onlyfor Comparison)

[0048] 1.2.1 Detection from Throat Swabs by Means of Cell Culture

[0049] The first throat swab in each case was immediately transferredafter collection to a tube containing 1.5 ml influenza virus transportmedium from the Virotest Company (Rosenbergstr. 85, D-70193 Stuttgart,Cat. No. 0500300) and cultured on MDCK cells (Madine-Darby Canine Kidneycells). The samples were cultured according to the methods described inthe literature (“Mikrobiologische Diagnostik”, Georg Thieme editor,Stuttgart, New York, 1992, publisher Friedrich Burkhard, page 371). Theresults are shown in table 1 in which “+” represents a positive resultand “−” represents a negative result.

[0050] 1.2.2 Detection from the Throat Swab by Means of an ImmunologicalRapid Test

[0051] The second swab was in each case examined immediately afterremoving the sample from the throat of the patient by means of animmunological antigen rapid test for the presence of influenza viruses(Influenza A/B Rapid Test from Roche Diagnostics GmbH, Mannheim,Germany, cat.—No. 2 158 663). The rapid test used in this casecorresponds essentially to the immunological rapid test described asexample 2 in EP-A 0 926 498. Reference is herewith expressly made tothis document.

[0052] In addition to the visual evaluation of the test strips, theintensity of the detection line after completion of the chromatographywas quantitatively measured by means of a remission photometricinstrument (ring illumination using 24 green LEDs at a wavelength of 555nm and CCD camera with a lens). The intensity of the detection linesignal was examined as a percentage of the remission (% rem; relative toa “white” area of the test strip which was assigned a remission of100%): remission values above 98.5% are detected as a negative signal bythe user; remission values between 96% and 98.5% are detected as aweakly positive signal; remission values of less than 96% are detectedas an unequivocally positive signal. The results are shown in table 1 inwhich “+” denotes a positive signal, “(+)” denotes a weakly positivesignal and “−” denotes a negative signal.

[0053] 1.3 Detection of Influenza A/B Viruses from Saliva

[0054] 1.3.1. Detection of Viral Nucleic Acid in the Saliva by Means ofRT-PCR

[0055] The saliva samples were examined by means of RT-PCR (reversedtranscriptase polymerase chain reaction) for the presence of viralinfluenza nucleic acid (in this case ribonucleic acid, RNA). Theanalytical process comprises the three conventional partial steps innucleic acid diagnostics of sample preparation (in order to separateinhibitors), amplification and detection of the nucleic acid (cf.1.3.1.a to 1.3.1.c in the following). The procedure was as follows:

[0056] 1.3.1.a Sample Preparation

[0057] The commercially available High Pure Viral RNA Kit from RocheMolecular Bio-chemicals (Sandhofer Straβe 116 in D-68305Mannheim/Germany) order No. 1 858 882 was used to isolate the viralinfluenza RNA. The procedure was according to the standard protocoldescribed in the product description by firstly binding 200 μl salivaper reaction vessel (filter tube) to the glass fleece of the filter tubein the presence of binding buffer and, after removing inhibitors thatmay be present by two wash steps, eluting the viral nucleic acid in 200μl elution buffer. In this process care was taken that the sample volumebefore (saliva) and after (eluate) the sample preparation was identicalso that no concentration of the influenza viruses had taken place. Ofcourse, it is also possible according to the invention to carry out theelution step with a smaller amount of elution buffer than the quantityof saliva and thus to concentrate or enrich the influenza viruses in theeluate.

[0058] 1.3.1.b RT-PCR

[0059] If not listed otherwise all reagents were obtained from RocheMolecular Biochemicals (see above).

[0060] The reaction volume per PCR reaction vessel was 50 μl. Thiscontained 10 μl sample volume (eluate from the sample preparation) and10 μl bicine buffer (5×RT-PCR buffer). The other components of themaster mix were present in the following final concentration: 2.5 mmol/lmanganese acetate, 0.2 mmol/l of each of the following dATP(2′-deoxyadenosine-5′ triphosphate), dCTP (2′deoxycytidine-5′triphosphate), dGTP (2′-deoxyguanosine-5′ triphosphate) and dUTP(2′-deoxyuridine-5 tri-phosphate) and 0.05 mmol/l dTTP(2′-deoxythymidine-5′ triphosphate); 0.01 U/μl UNG (uracil DNAglycosylase); 0.2 U/μl Tth polymerase from Thermus thermophilus HB8; 0.8U/μl RNase inhibitor and 1.0 μmol/l of forward and reverse primer.

[0061] The sequence of the primers was obtained from the literature(James C. Donofrio et al., Detection of Influenza A and B in RespiratorySecretions with the PCR, 1992, PCR Methods and Applications 1, page263-268). The primers are type-specific for influenza A and have ahighly conserved gene segment (position 101 to 312) of the matrix geneof 212 base pairs in length. The reverse primer was labelled at the 5′end with biotin for subsequent detection of the amplification product inthe microtitre plate. Type-specific primers for influenza B are alsoknown in the literature (James C. Donofrio et al., Detection ofInfluenza A and B in Respiratory Secretions with the PCR, 1992, PCRMethods and Applications 1, page 263-268).

[0062] The master mix was amplified in a Perkin Elmer 9600 thermocyclerusing the following temperature profile: 20 minutes room temperatureafter addition of UNG+45 minutes 60° C.+2 minutes 94° C.+10 cycles (30seconds 94° C.+60 seconds 50° C.+90 seconds 68° C.)+35 cycles (30seconds 94° C.+60 seconds 60° C.+90 seconds 68° C.)+7 minutes 68° C.

[0063] 1.3.1c Hybridization and Detection

[0064] The nucleic acid amplified by the RT-PCR was detected using thePCR ELISA (DIG detection) from Roche Molecular Biochemicals cat. No. 1636 111. With the exception of the pipetting volumes, all steps werecarried out according to the package insert.

[0065] 10 μl of the amplification product was mixed with 20 μldenaturating solution in a 1.5 ml reaction vessel and incubated for 10minutes at room temperature. Afterwards 250 μl of a digoxigenin-labelledhybridization probe was added. The concentration of the hybridizationprobe was 70 ng/ml.

[0066] The sequence of the hybridization probe was obtained from theliterature (James C. Donofrio et al., Detection of Influenza A and B inRespiratory Secretions with the PCR, 1992, PCR Methods and Applications1, page 263-268). The probe is directed against position 177-205 of thematrix gene (segment 7) of influenza A. Corresponding probes forinfluenza B are also known from the literature (James C. Donofrio etal., Detection of Influenza A and B in Respiratory Secretions with thePCR, 1992, PCR Methods and Applications 1, page 263-268).

[0067] 200 μl of the reaction mixture was transferred to a well of astreptavidin-coated microtitre plate and incubated on a shaker for 1hour at 37° C. After binding the hybridization product to thestreptavidin of the microtitre plate wall, the contents of the wellswere aspirated and washed three times with 300 μl wash solution eachtime. Afterwards 200 μl anti-digoxigenin-peroxidase substrate(anti-DIG-POD conjugate) was added to the well and the solution wasincubated for 30 minutes at 37° C. on a shaker. After binding the PODconjugate the contents of the wells were aspirated, washed 3 times with300 μl wash solution and subsequently 200 μl2,2′-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium saltsubstrate (ABTS substrate) was added. After a final incubation for 30minutes at 37° C., the colour development at 405 nm was measured on amicrotitre plate reader.

[0068] For each PCR determination of saliva samples, several negativecontrols (DEMC water (article No. 16-001Y “Accu Gene Water, molecularbiology grade, autoclaved” manufactured by Bio Whittaker Europe S.p.r.1.(Parc Industriel de Petit-Rechain, B-4800 Verviers, Belgium) werecarried out in parallel to exclude false-positive values bycontamination and several positive controls (dilution series of MDCKculture supernatant containing influenza viruses) were determined inparallel by sample preparation and RT-PCR. In addition an internal kitpositive control was also carried out in order to check the detectionreagents on the microtitre plate.

[0069] The absorbance values of the parallel negative controls in themicrotitre plate were usually 100-150 mA. The values of the internalpositive control of the kit that was carried out in parallel wereusually 1000-1500 mA.

[0070] Absorbance values of ≧300 mA were defined as a positive signalfor patient samples which corresponds to a signal that was more thantwice the blank or zero value. The results are shown in table 1 in which“+” denotes a positive result and “−” denotes a negative result.

[0071] 1.3.2. Detection of a Viral Antigen in Saliva by Means of anImmunological Rapid Test

[0072] The influenza A/B rapid test from Roche Diagnostics GmbH, cat.No. 2 158 663 was used to detect influenza A/B viruses in saliva. Therapid test that was used corresponds essentially to the immunologicalrapid test described as example 2 in EP-A 0 926 498. Reference isexpressly made herewith to this document.

[0073] The test is usually used to type-specifically detect viralnucleoprotein in throat swabs by means of an immunologicalchromatographic test strip. The test does not differentiate betweeninfluenza A and influenza B viruses.

[0074] In order to ensure a good chromatography of the test strip usingsaliva as the sample material, a special lysing/elution buffer was usedwhich is not a component of the test kit. The special lysing/elutionbuffer has the following composition:

[0075] 0.9% by weight NaCl, 2 mM KH₂PO₄, 10 mM Na₂HPO₄, 0.095% by weightNaN₃, 10 mM EDTA, 1.5% by weight bovine serum albumin (BSA), 1.5% byweight Triton® X-100.

[0076] The test was carried out as follows:

[0077] Instead of firstly eluting a throat swab as the patient samplewith 3 portions (=800 μl) of the lysing/elution buffer contained in thetest kit as described in the package insert, 300 μl saliva and 500 μl ofthe special lysing/elution buffer were added to a reaction vessel andmixed.

[0078] The remaining test procedure was carried out according to thesteps described in the package insert. This comprises firstly adding twodrops of antibody solution 1 (contains biotinylated monoclonalantibodies against nucleoprotein A and nucleoprotein B), adding twodrops of antibody solution 2 (contains digoxigenylated monoclonalantibodies against nucleoprotein A and nucleoprotein B) and subsequentlychromatographing this reaction mixture on a test strip.

[0079] As known from EP-A 0 926 498 the test strip contains a conjugatefleece which is reversibly impregnated with a gold conjugate that can bedetached in the sample liquid. The gold particles are adsorptivelycoated with a monoclonal antibody against digoxigenin.

[0080] A nitrocellulose membrane on which polystreptavidin as adetection line and a polyclonal antibody PAB<mouse Fcγ>S-IgG as acontrol line are irreversibly impregnated is located in another zone ofthe test strip downstream in the direction of chromatography.

[0081] The sandwich complex formed in the reaction vessel in thepresence of the analyte comprising biotinylatedantibody/nuleoprotein/digoxigenylated antibody chromatographs across thetest strip, binds the gold conjugate after it has been solubilized viathe anti-digoxigenin antibody to the digoxigenin-labelled anti-influenzaantibody of the sandwich complex and is subsequently captured on thepolystreptavidin line on the nitrocellulose membrane by means of thebiotin-labelled anti-influenza antibody of the sandwich complex. As aresult a red line becomes visible on the nitrocellulose membrane whichrepresents a positive test signal.

[0082] Excess gold conjugate chromatographs downstream and is capturedon the control line of the nitrocellulose membrane as another visiblered line by means of PAB<mouse Fcγ>S-IgG.

[0083] In addition to the visual evaluation of the test strips, theintensity of the detection line after completion of the chromatographywas quantitatively measured by means of a remission photometricinstrument (ring illumination using 24 green LEDs at a wavelength of 555nm and CCD camera with a lens). The intensity of the detection linesignal was examined as a percentage of the remission (% rem; relative toa “white” area of the test strip which was assigned a remission of100%): remission values above 98.5% are detected as a negative signal bythe user; remission values between 96% and 98.5% are detected as aweakly positive signal; remission values of less than 96% are detectedas an unequivocally positive signal. The results are shown in table 1 inwhich “+” denotes a positive signal, “(+)” denotes a weakly positivesignal and “−” denotes a negative signal.

[0084] 1.4. Results

[0085] Some results obtained with patient samples are shown in thefollowing as an example (table 1).

[0086] These results relate exclusively to the detection of influenza Aviruses since only influenza A was predominant at the time of samplecollection and no patient samples with influenza B were found. Expertsknow that influenza B, in contrast to influenza A, does not circulate inevery winter season and even if both influenza A/B viruses occur in awinter season, influenza B is always much less prevalent.

[0087] It should also be mentioned that artificially prepared influenzaB-positive saliva samples (pooled saliva samples which have been spikedwith a culture supernatant of influenza B viruses) were also suitablefor detecting influenza viruses in saliva in the nucleic acid test aswell as in the immunological test. The results shown do not thereforerepresent a limitation in the sense of the invention with regard toseparate or joint detection of influenza B viruses in addition toinfluenza A viruses from saliva. Rather saliva is suitable as a samplematerial for the detection of influenza A/B viruses. TABLE 1 Throat swabsamples (only for comparison) Saliva samples Per- immunologicalimmunological son rapid test PCR rapid test No.^(A) culture^(B)visual^(C) photometric^(D) result^(E) visual^(C) photometric^(D)1 + + + + + + 2 + (+) (+) + + + 3 + (+) (+) + + + 4 + + + + + +5 + + + + + + 6 + (+) (+) + + + 7 + − − + (+) (+) 8 + − − + (+) (+) 9 −− − − − − 10  − − − − − − 11  − − − − − − 12  − − − − − − 13  − − − − −− 14  − − − − − −

[0088] The following is apparent from the results shown in table 1:

[0089] Not all persons (No. 1-10) classified as influenza patientsaccording to the clinical symptoms were in fact influenza positive.Persons No. 9 and 10 were negative in the culture of the throat swab aswell as in the PCR of the saliva as well as in the rapid test of thesaliva and throat swab. This emphasizes the statement made in theintroduction and known among experts that it is not possible to make adefinitive diagnosis for influenza solely on the basis of clinicalsymptoms.

[0090] The throat swabs and saliva samples of asymptomatic persons (No.11-14) were negative with all listed test methods which demonstrates theclinical specificity of these methods.

[0091] Of the eight persons found to be influenza A positive by means ofcell culture of throat swabs (No. 1-8; so-called culture-positivepersons), six persons were also diagnosed as positive with theimmunological rapid test using throat swabs. This illustrates that theclinical sensitivity of these previously available rapid tests usingswabs as a sample material is less than that of the cell culture whichis recognized at present as the gold standard (clinical sensitivities ofvarious immunological rapid tests according to the package inserts ofthe respective manufacturers: Quidel 73% for nose swabs, Roche 70% forthroat swabs, Biostar 60% for throat swabs and 83% for nasopharyngealswabs).

[0092] In contrast the corresponding saliva samples from all eightculture-positive patients were found to be positive with theimmunological rapid test.

[0093] Furthermore the comparison of the remission values (not shown inthe table) shows that the intensities of the detection line on theimmunological rapid test strips using saliva samples were somewhat moreintensive than the corresponding intensities of the detection line whenusing throat swabs.

[0094] The results of the rapid tests of saliva are thus in agreementwith the corresponding PCR results from saliva and demonstrate thatinfluenza A/B viruses can be reliably detected in saliva as a samplematerial even without prior enrichment during the sample collection.

1. Method for the detection of an infection with the influenza A and/orinfluenza B virus comprising the steps i) obtaining a saliva sample, ii)preparing the saliva sample for the detection and iii) detecting theinfluenza A and/or influenza B virus in the saliva sample.
 2. Method asclaimed in claim 1, characterized in that the saliva sample which isobtained in step i) is spontaneously formed saliva.
 3. Method as claimedin claim 1, characterized in that the saliva sample in step i) isobtained with a saliva collection device without virus-specificenrichment.
 4. Method as claimed in one of the claims 1 to 3,characterized in that viral influenza A and/or influenza B RNA isisolated from the saliva sample in step ii), amplified by RT-PCR anddetected in step iii).
 5. Method as claimed in one of the claims 1 to 3,characterized in that in step iii) the saliva sample is examinedimmunologically for the presence of influenza A and/or influenza Bviruses.
 6. Method as claimed in claim 5, characterized in that thesaliva sample is treated in step ii) with a lysing buffer such thatviral nucleoprotein of the influenza A and/or influenza B virus isreleased and this nucleoprotein is immunologically detected in stepiii).
 7. Test kit for the detection of an infection with the influenza Aand/or influenza B virus containing i) a device for collecting a salivasample and ii) reagents and auxiliary agents for the detection ofinfluenza A and/or influenza B viruses.
 8. Use of saliva as the samplematerial for the detection of an infection with the influenza A and/orinfluenza B virus.
 9. Use of saliva as claimed in claim 8, characterizedin that the saliva is spontaneously formed saliva.
 10. Use of saliva asclaimed in claim 8, characterized in that the saliva sample is collectedwith a saliva collecting device without virus-specific enrichment. 11.Use of saliva as claimed in one of the claims 8 to 10, characterized inthat an infection with influenza A and/or influenza B viruses isdetected by means of an immunological test.
 12. Use of saliva as claimedin one of the claims 8 to 10, characterized in that an infection withinfluenza A and/or influenza B viruses is detected by means of a nucleicacid detection.